Recognition of E. coli tryptophan synthase by single-chain Fv fragments: comparison of PCR-cloning variants with the parental antibodies.
The use of a recombinant antibody fragment instead of a complete antibody, as a conformational probe for protein structure and folding studies, can be technically advantageous provided that the recombinant fragment and its parental antibody recognize the antigen through the same mechanism. Monoclonal antibodies mAb19 and mAb93 are directed against the TrpB2 subunit of Escherichia coli tryptophan synthase and they have been extensively used as conformational probes of this protein. DNA sequences coding for single-chain variable fragments (scFv) of mAb19 and mAb93 were cloned and assembled by reverse transcription of the mRNAs from hybridomas and PCR amplification. A specialized plasmid vector, pFBX, was constructed; it enabled to express the scFvs as hybrids with the maltose-binding protein (MalE) in E. coli, and to purify them by affinity chromatography on cross-linked amylose. Six independent clones were sequenced for each hybridoma. All of them had differences in their nucleotide and amino acid sequences. A competition ELISA and the BIAcore biosensor apparatus were used to compare the energetics and kinetics with which the parental antibodies and the hybrids bound TrpB2. The antigen binding properties of the hybrids were close to those of the parental antibodies and they were only weakly affected by the differences of sequence between the clones, with one exception. The stability of one of the hybrids and its antigen binding properties were strongly modified by a change of Gln6 into Glu, introduced into its VH domain by the PCR primers. Simple models of bimolecular interaction did not fully account for the kinetic profiles obtained with the parental antibodies and the hybrids, and this complexity suggested the existence of a conformational heterogeneity in these molecules.
Brégégère
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F
Unité de Biochimie Cellulaire (CNRS URA 1129), Institut Pasteur, Paris, France.
England
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P
Djavadi-Ohaniance
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L
Bedouelle
H
H
D018528:ATP-Binding Cassette Transporters; D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000737:chemistry; D001426:Bacterial Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D001483:Base Sequence; D002352:Carrier Proteins / Q000737:chemistry; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D007128:Immunoglobulin Fragments / Q000737:chemistry* / Q000276:immunology; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000276:immunology; D008320:Maltose / Q000378:metabolism; D058619:Maltose-Binding Proteins; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D033902:Periplasmic Binding Proteins; D016133:Polymerase Chain Reaction; D017510:Protein Folding; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000276:immunology; D014367:Tryptophan Synthase / Q000737:chemistry* / Q000276:immunology
D018528:ATP-Binding Cassette Transporters; D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; C461619:MalE protein, E coli; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D033902:Periplasmic Binding Proteins; D011994:Recombinant Proteins; C055966:immunoglobulin Fv; C461599:maltose transport system, E coli; D008320:Maltose; D014367:Tryptophan Synthase